Our long-term objective is to improve therapies for progressive pulmonary hypertension (PH) in infants suffering from chronic cardiopulmonary disorders associated with persistent or episodic hypoxia. To do this, we developed a model of PH in newborn pigs. We have shown that PH develops within 3 days exposure to hypoxia and worsens when hypoxic exposure is extended to 10 days. The progressive development of PH in newborn pigs is accompanied by NO signaling impairments, including eNOS uncoupling. These findings serve as the basis for testing the hypothesis that restoring eNOS uncoupling will improve NO production and ameliorate PH. Our experimental design will test the hypothesis that combined treatment with the L-arginine- NO precursor L-citrulline, plus the co-factor BH4, restores pulmonary vascular NO signaling and ameliorates PH more effectively than either treatment alone, by more effectively recoupling eNOS, shifting the balance of eNOS-generated free radicals away from superoxide and toward NO production. The aims of this proposal are to: 1) evaluate the mechanisms by which combined therapy with L-citrulline and BH4 increase pulmonary vascular NO production 2) establish the efficacy and safety of combined vs sole therapy with oral L-citrulline or BH4 to ameliorate chronic hypoxia-induced PH. Studies will evaluate the ability of these therapies to arrest the progressive development of PH when given chronically to piglets exposed to hypoxia and to acutely improve pulmonary vascular responses to NO when administered to piglets with PH. As part of the first aim, studies will address current gaps in our knowledge about the oxidation and loss of BH4 in the neonatal pulmonary circulation. This will include delineating the sources of superoxide, including NADPH oxidase family enzymes, that contribute to oxidation of BH4, and, hence, uncoupled eNOS. As part of the second aim, we will determine if combined therapy with L-citrulline and BH4 improves the parameters of NO signaling that are perturbed with chronic hypoxia. These studies will provide invaluable information about restoring impaired NO signaling pathways that can ultimately be translated into important clinical trials to treat infants with chronic cardiopulmonary conditions and PH due in part to hypoxia.